RD21-like proteases: key effector hubs in plant-pathogen interactions.

Over the past decades, numerous studies have demonstrated that proteases serve as a crucial regulatory mechanism in controlling plant immunity. In this review, we specifically focus on the role of one subfamily of RD21-like papain-like cysteine proteases that carry a C-terminal granulin domain. These proteases share high homology but have been described under very different names in different plant species.

Subtilase SBT5.2 inactivates flagellin immunogenicity in the plant apoplast.

Most angiosperm plants recognise the 22-residue flagellin (flg22) epitope in bacterial flagellin via homologs of cell surface receptor FLS2 (flagellin sensitive-2) and mount pattern-triggered immune responses. However, flg22 is buried within the flagellin protein indicating that proteases might be required for flg22 release. Here, we demonstrate the extracellular subtilase SBT5.

Novel structural insights at the extracellular plant-pathogen interface.

Plant pathogens represent a critical threat to global agriculture and food security, particularly under the pressures of climate change and reduced agrochemical use. Most plant pathogens initially colonize the extracellular space or apoplast and understanding the host-pathogen interactions that occur here is vital for engineering sustainable disease resistance in crops. Structural biology has played important roles in elucidating molecular mechanisms underpinning plant-pathogen interactions but only few studies have reported structures of extracellular complexes.

Using AlphaFold Multimer to discover interkingdom protein-protein interactions.

Structural prediction by artificial intelligence can be powerful new instruments to discover novel protein-protein interactions, but the community still grapples with the implementation, opportunities and limitations. Here, we discuss and re-analyse our in silico screen for novel pathogen-secreted inhibitors of immune hydrolases to illustrate the power and limitations of structural predictions. We discuss strategies of curating sequences, including controls, and reusing sequence alignments and highlight important limitations caused by different platforms, sequence depth and computing times.

Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors.

Secreted immune proteases "Required for Cladosporium resistance-3" (Rcr3) and "Phytophthora-inhibited protease-1" (Pip1) of tomato (Solanum lycopersicum) are both inhibited by Avirulence-2 (Avr2) from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signaling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity.

The proteome of Nicotiana benthamiana is shaped by extensive protein processing.

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel.

Improving transient protein expression in agroinfiltrated Nicotiana benthamiana.

Agroinfiltration of Nicotiana benthamiana is routinely used in plant science and molecular pharming to transiently express proteins of interest. Here, we discuss four phenomena that should be avoided to improve transient expression. Immune responses can be avoided by depleting immune receptors and employing pathogen-derived effectors; transcript degradation by using silencing inhibitors or RNA interference machinery mutants; endoplasmic reticulum stress by co-expressing chaperones; and protein degradation can be avoided with subcellular targeting, protease mutants and co-expressing protease inhibitors.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity.

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins-PsbO, PsbP, and PsbQ-integral components of photosystem II's oxygen-evolving complex.

Proteolysis in plant immunity.

Compared with transcription and translation, protein degradation machineries can act faster and be targeted to different subcellular compartments, enabling immediate regulation of signaling events. It is therefore not surprising that proteolysis has been used extensively to control homeostasis of key regulators in different biological processes and pathways. Over the past decades, numerous studies have shown that proteolysis, where proteins are broken down to peptides or amino acids through ubiquitin-mediated degradation systems and proteases, is a key regulatory mechanism to control plant immunity output.

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes.

Role of glutathione S-transferases in the mode of action of herbicides that inhibit amino acid synthesis in Amaranthus palmeri.

Acetolactate synthase inhibitors (ALS inhibitors) and glyphosate are two classes of herbicides that act by inhibiting an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. Besides amino acid synthesis inhibition, both herbicides trigger similar physiological effects in plants. The main aim of this study was to evaluate the role of glutathione metabolism, with special emphasis on glutathione S-transferases (GSTs), in the mode of action of glyphosate and ALS inhibitors in Amaranthus palmeri.

Releasing hidden MAMPs from precursor proteins in plants.

The recognition of pathogens by plants at the cell surface is crucial for activating plant immunity. Plants employ pattern recognition receptors (PRRs) to detect microbe-associated molecular patterns (MAMPs). However, our knowledge of the release of peptide MAMPs from their precursor proteins is very limited.

Glutathione Transferase Photoaffinity Labeling Displays GST Induction by Safeners and Pathogen Infection.

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in the detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. In this study, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study GSTs globally in various plant species. The small-molecule probe contains glutathione, a photoreactive group and a minitag for coupling to reporter tags via click chemistry.

Cysteine proteases are activated in sensitive Amaranthus palmeri populations upon treatment with herbicides inhibiting amino acid biosynthesis.

The herbicides glyphosate and pyrithiobac inhibit the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid biosynthetic pathway and acetolactate synthase (ALS) in the branched-chain amino acid biosynthetic pathway, respectively. Here we characterise the protease activity profiles of a sensitive (S), a glyphosate-resistant (GR) and a multiple-resistant (MR) population of Amaranthus palmeri in response to glyphosate and pyrithiobac. Amino acid accumulation and cysteine protease activities were induced with both herbicides in the S population and with pyrithiobac in the GR population, suggesting that the increase in cysteine proteases is responsible for the increased degradation of the available proteins and the observed increase in free amino acids.

AlphaFold-Multimer predicts cross-kingdom interactions at the plant-pathogen interface.

Adapted plant pathogens from various microbial kingdoms produce hundreds of unrelated small secreted proteins (SSPs) with elusive roles. Here, we used AlphaFold-Multimer (AFM) to screen 1879 SSPs of seven tomato pathogens for interacting with six defence-related hydrolases of tomato. This screen of 11,274 protein pairs identified 15 non-annotated SSPs that are predicted to obstruct the active site of chitinases and proteases with an intrinsic fold.

Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Epoxide Hydrolase Related to Phytohormone Signaling.

Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in that revealed Neratinib (), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that covalently modifies a surface-exposed cysteine residue of epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition.

The root pathogen secretes modular proteases in pea apoplast during host infection.

To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete , which causes root rot diseases on legumes.

Depletion of the NbCORE receptor drastically improves agroinfiltration productivity in older Nicotiana benthamiana plants.

Nicotiana benthamiana is increasingly used for transient gene expression to produce antibodies, vaccines, and other pharmaceutical proteins but transient gene expression is low in fully developed, 6-8-week old plants. This low gene expression is thought to be caused by the perception of the cold shock protein (CSP) of Agrobacterium tumefaciens. The CSP receptor is contested because both NbCSPR and NbCORE have been claimed to perceive CSP.

Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface.

The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae.

Activity-based probes trap early active intermediates during metacaspase activation.

Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors . By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium.